Numerous techniques exist for the molecular separation of the components of a given sample for either analysis purposes or for product preparation purposes. One type of molecular separation embraces a variety of processes for effecting differential distribution of the sample components between two phases and such processes are generally referred to as chromatography. The differential distribution is achieved by an interchange between a moving phase, which can be a liquid or gas, and a stationary phase.
1. Field of the Invention
This invention relates to novel molecular separation columns, e.g. chromatography columns, and more particularly to a novel stationary phase for use in such columns.
Chromatography is a general term applied to a wide variety of separation techniques based upon the sample interchange between a moving phase, which can be a gas or liquid, and a stationary phase. When gas is the moving phase (or "mobile phase" as referred to in chromatographic terminology), the technique is termed gas chromatography and when liquid is the mobile phase, the technique is termed liquid chromatography.
The so-called "chromatographic adsorption method" of analysis was originated by the Russian botanist, M. Tswett, Ber. Deut. Botan. Ges., 24, 316, 1906, who used it for separating components of plant pigments. Little notice of this work was taken until 1931 and up to 1940 the emphasis was on frontal and displacement analysis. The theory of chromatography was originated by Wilson, J. N., J. Amer. Chem. Soc., 62, 1583, in 1940. Although the important historical role of this work has been largely neglected because the quantitative theory did not allow for diffusion or nonequilibrium between the mobile and the stationary phases, Wilson offered a sound qualitative description of nonequilibrium and its important place in chromatography. He also explained the role of longitudinal diffusion. Although the large spreading effects arising from low adsorption and desorption rates can be diminished by decreasing the flow rates, this in turn gives rise to spreading effects due to large longitudinal diffusion effects.
Following Wilson's work the Nobel prize wining paper of A. J. D. Martin and R. L. M. Synge, Biochem. J., 35, 1358, 1941, appeared which introduced the plate theory of chromatography and revolutionized liquid chromatography. They also suggested using a gas as the mobile phase. Gas chromatography was first tried by A. T. James and A. J. P. Martin, Biochem. J., 50, 6979, in 1952. Since then, however, voluminous literature has been published dealing with gas-liquid and gas-solid chromatography, and gas chromatography has evolved into a sophisticated analytical technique.
Initially, liquid chromatography was performed in large diameter glass columns under normal pressure. These conditions led to long analysis times and a generally tedious procedure. However in recent years, with the introduction of high pressure pumps, advances in both instrumentation and column packings have occurred so rapidly that it has become difficult to keep pace with the amount of literature that is being published, and liquid chromatography is rapidly gaining ground on gas chromatography in becoming of equal stature.
Separations can be classified into either analytical or preparative depending on the objective. In analytical separations, the objective is high resolution separation and identification and quantification of the various components of a sample mixture. In preparative chromatography, on the other hand, the objective is the isolation of pure quantities of the desired constituents in the sample. Liquid chromatography is advantageous over gas chromatography in that the former can be both an excellent analytical as well as a preparative technique. Gas chromatography is very limited in its application as a preparative tool because of the very small sample sizes. In liquid chromatography, on the other hand, milligram to gram quantities of preparative separations are not uncommon depending on the chromatographic column diameter and the amount of stationary phase.
The collection of chromatographic techniques can be classified in several ways and the most fundamental is based on naming the types of phases used. Liquid adsorption chromatography is used extensively for organic and biochemical analysis but is limited because there are only a few suitable adsorbents. The distribution coefficient of adsorption often depends on total concentraton and this behavior often results in incomplete separations. Gas-solid chromatography has generally suffered from the same defects as liquid adsorption chromatography. Ion exchange chromatography is a special field of liquid-solid chromatography and is specifically applicable to ionic species. Affinity chromatography is based on the attraction (affinity) of a ligand bonded to the solid stationary phase for a given component of the sample. Liquid-liquid or partition chromatography involves the use of a thin layer of liquid held in place on the surface of a porous inert solid as the stationary phase. Paper chromatography is a special field of liquid-liquid chromatography in which the stationary liquid is a film of water adsorbed on a paper mat and thin layer chromatography is similar to paper chromatography except that the paper is replaced by a glass or plastic plate coated with a thin layer of alumina, silica gel or other powdered material.
Column efficiency is generally measured in terms of H, sometimes referred to HETP (height equivalent to a theoretical plate), which is the column length divided by the total number of theoretical plates (n) contained in that length. H is generally considered to be a summation of three contributions, i.e. the contribution from non-equal paths (eddy diffusion), the contribution from diffusion along the column (longitudinal diffusion) and the contribution from non-equilibrium (mass transfer). The eddy diffusion is directly proportional to the diameter of the particles constituting the stationary phase. The less homogeneous the structure, the larger is the contribution from non-equal paths. Conventional chromatographic theory thus predicts that finer packing geometries will have decreased diffusional boundary layers, i.e. shorter paths for material transport to the solid surface which will result in increased efficiency. Current chromatographic theory, and therefore current practice, leads to the use of very fine, homogeneous spherical packing. A complicating factor, however, is that the resistance of the column to fluid flow, i.e. the pressure drop across the column, is inversely proportional to the square of the diameter of the particle. Therefore, halving the particle diameter will increase the pressure drop by a factor of 4.
Additionally, as known in the art, gel substrates are unable to withstand significant pressure drops and/or low flow rates.
2. Prior Art
The use of adsorbents or particulates carried by fibers or paper has been used in the filter art for some time, see, for example, the following U.S. Pat. Nos.:
2,143,044 to Wicks et al; PA1 2,746,608 to Briggs; PA1 3,238,056 to Pall et al; PA1 3,253,978 to Bodendorf et al; PA1 3,591,010 to Pall et al; PA1 4,007,113 to Ostreicher; PA1 4,160,059 to Samejima; and PA1 4,238,334 to Halbfoster. PA1 U.S. Pat. No. 3,570,673 to Dutzetal; PA1 U.S. Pat. No. 3,307,333 to Norem et al; PA1 U.S. Pat. No. 4,169,790 to Pretorius et al; and PA1 U.S. Pat. No. 4,070,287 to Wiegand.
Carbon has been loaded on a sheet having particles homogeneously distributed and firmly retained therein, as described in U.S. Pat. No. 3,149,023 to Bodendorf et al. The Bodendorf et al sheets are used as cigarette filters, air filters, gas filters, wrappers for fruit and substances prone to discoloration or spoilage by gases in the atmosphere deodorizer layers in laminated sheet products for sanitary napkins, and for surgical dressings for wounds, and the like.
Somewhat similar sheets, as described in the aforementioned patents, have been employed in paper and thin layer chromatography.
Malcolm, U.S. Pat. No. 3,647,684 teaches a thin layer chromatography medium which takes the form of a self-supporting flexible sheet structure having a major proportion of a chromatographic adsorbent such as silicic acid uniformly and homogeneously dispersed with a minor amount of structurally stabilizing inorganic fibers such as glass fibers disposed in a randomly oriented network of a cationic material such as cationic starch.
Leifield, U.S. Pat. No. 3,455,818 teaches sorbent sheets useful for chromatography carried out in the same general manner as conventional thin layer or paper chromatography. The sheets are prepared by dispersing fine fibers of a non-cellulosic material such as fibrous glass together with a high proportion of the desired powdered sorbent in a suitable liquid medium which is flowed onto a porous support followed by removing the liquid. The sheets can be used in a column by rolling one or several of the sheets into a compact roll and inserting it into a glass tube or cylinder such that the interface between sheet surfaces is parallel to and in the path of the mobile phase flow.
Fibers or filament type packings for molecular separation columns have been used, see for example, Miller et al, The Use Of Textile Yarns In Separation Processes, Textile Research Journal, January, 1980, pp. 10 et seq.; Brown et al, Macroreticular Resin Columns. I. Model of Bend and Filament Packings, Separation Science and Technology, 15(a), pp. 1533-1553 (1980); and Partridge, Nature, 1123-1125 (March 18, 1967). Other references which suggest the use of fibers for molecular separation packing are:
It is generally accepted in the art that scaling up from laboratory results is difficult, particularly in chromatographic processes where theoretical models are unsatisfactory. The construction of commercial installations based upon knowledge gained from laboratory experiments in this field has turned out to be a major problem. According to those skilled in the art, the use of large columns of resins, for example organic gel columms, is not desirable because of compaction, poor separation results and because of excessive dilution of the eluted components, both of which factors make the process an uneconomical one. If a liquid is introduced evenly across the top of the column, a portion of the front thereof moves downwardly at a rate different from the rate of movement of the balance of the liquid, running obliquely, causing "tailing" and "finger formation" to occur in the bed. To avoid these problems, it is desirable that the front or leading edge of each liquid or eluent, supplied to the top of the column, move downwardly at a uniform rate, the front remaining substantially in a narrow band lying in a horizontal plane.
These prior art problems are discussed, for example, by Baddour in U.S. Pat. No. 3,250,058. Good separations are achieved using thin laboratory columns, but when attempts are made to repeat the separation on a technical or commercial scale, using columns of 5 cm or more in diameter, it is found that "tailing" and "finger formation" occur in the column, both of which causes dilution and poor separation results. Baddour attempts to overcome these problems by the introduction of an arrangement of transverse baffles within the column to induce lateral flow of the liquid flowing through the column. In addition, Baddour finds it necessary to use these baffles in combination with lateral baffles.
The idea of a forced vertical flow in large scale columns was further developed by Lauer et al. in U.S. Pat. No. 3,539,505, who introduced units for radial mixing into the column or divided the column into several short sections as described in German Patent Application DOS No. 2,036,525. Yet another approach to the problem is described in the German Patent Application DOS No. 2,224,794 and Japanese Patent Specification No. 73-68752, according to which the column is saturated with the solution which is to be separated. By means of the saturated columns and countercurrent flow, the disturbances caused by density gradients in the column are avoided.
The rather complicated methods which are described above make it possible to conduct large scale chromatographic separation procedures on a commercial basis. However, these methods lead to complicated column structures and to methods which are difficult to accomplish on a commercial scale. Where there are built-in structures within the column, substantial problems occur, for example, when the resin is backwashed. Backwashing is required in these procedures after a certain number of cycles because mechanical impurities from the feed or eluent accumulate on the resin bed so that the performance of the column gradually decreases in efficiency. It is obvious that built-in structures in the column are a nuisance in such situations. The ideas of saturated resin beds and countercurrent flow also lead to complicated structures as described in the German Patent Application DOS No. 2,224,794 or to a complicated procedure of operating the system.
Huber describes another approach in his U.S. Patent No. 3,856,681 where elongated rod-like elements were arranged parallel to the axis of the column but those elements produced unsymmetrical column cross-sections causing difficult column packing and uneven fluid flow and also limited overall productive output of the column.
Huber, in his U.S. Pat. No. 3,856,681 attempts to obtain uniform flow across a preparative or production chromatography column through the use of a plurality of layers of chromatographic media arranged adjacent to each other, with the thickness dimension of the layers extending substantially perpendicular to the primary fluid flow axis and preferably spaced laterally from each other by relatively inert partitioning means interposed between the layers. If desired, relatively large particles of chromatographic media or relatively inert material can be uniformly distributed through the chromatographic media layer to reduce the overall pressure drop through the final column. The surface of the chromatographic medium is parallel to and in the same direction as the mobile phase fluid flow.
McDonald et al in U.S. Pat. No. 4,211,656 describes a cartridge which triaxially compresses the particulate packing material to assure even flow through the column.
It has now been discovered that a column in which a mobile phase flows through a solid stationary phase can be constructed in direct contradiction to conventional chromatography packing theory if the stationary phase "system" is, broadly, a body of particulate immobilized in a porous matrix of fiber. This new stationary phase has the advantage of both low pressure drop and low diffusion resistance making it particularly suitable for commercial scale separations, particularly liquid separations. Baffle arrangements are unnecessary. As a result, it is possible to construct stable, high flow separation columns of high capacity and shorter run times which have good pressure response, freedom from channeling or fluid bypass, ease of regeneration to reproducible reuse, and the capacity to be shipped under ambient conditions or stored indefinitely. Additionally, the edges of the new stationary phase cooperate with the interior wall of the separation column to form a substantially fluid tight seal therewith, thus preventing channeling near the walls.